Investigators in the Section on Metabolic Regulation have focused on the following projects: (i) Mechanistic studies of Cu,Zn-superoxide dismutase (SOD1) mutant-mediated familial amyotrophic lateral sclerosis (FALS). Amyotrophic lateral sclerosis (ALS) is a fatal degenerative disease of motor neurons in the cortex, brainstem, and spinal cord. Missense mutations of SOD1 are linked to familial amyotrophic lateral sclerosis through a yet-to-be identified toxic-gain-of-function. One of the proposed mechanisms involves enhanced aggregate formation, which is consistent with the appearance of mutant SOD1-containing inclusions in the spinal cord. However, a recent study showed that dual transgenic mice that overexpressed both G93A and CCS copper chaperone (G93A/CCS) do not exhibit SOD1-positive aggregates yet show accelerated FALS symptoms with enhanced mitochondrial pathology and G93A mitochondrial translocation compared to G93A mice (PNAS 104, 6072-6077, 2007). Using a dicistronic mRNA to simultaneously generate hSOD1 mutants, G93A, A4V, and G85R, and hCCS in AAV293 cells, we revealed: (i) for active SOD1 mutants, via its copper chaperone activity, CCS prevents accumulation of SOD1 mutant aggregates by catalyzing the formation of active and soluble SOD1 homodimers. With inactive G85R, via its novel copper chaperone-independent molecular chaperone activity, CCS forms a stable hetrodimer with G85R. Like CCS itself, this hetrodimer is readily degraded via a macroautophagy-mediated pathway to prevent aggregate accumulation;(ii) CCS facilitates mitochondrial translocation of inactive SOD1 mutants under oxidative stress conditions, e.g. hydrogen peroxide treatment. These results, together with previous reports on the enhanced free radical-generating activity of FALS SOD1 mutants, provide a mechanistic explanation for the observations with G93A/CCS dual transgenic mice and suggest that free radical generation by FALS SOD1, enhanced by CCS, may, in part, be responsible for the FALS SOD1 mutant-linked aggregation, mitochondrial translocation, and degradation. (ii) Regulation of 2-cys-peroxiredoxin I, a dual-function enzyme, by glutathionylation. Reversible protein glutathionylation, a redox-sensitive regulatory mechanism, plays a key role in cellular regulation and cell signaling. Peroxiredoxins (Prxs), a family of peroxidases that is involved in removing hydrogen peroxide and organic hydroperoxides, are known to undergo a functional change from peroxidase to molecular chaperone upon overoxidation of its catalytic cysteine. The functional change is caused by a structural change from low molecular weight oligomers to high molecular weight complexes that possess molecular chaperone activity. We reported earlier that Prx I can be glutathionylated at three of its cysteine residues, Cys52, 83, and 173 (JBC 284, 23364, 2009). In the current study, using analytical ultracentrifugation analysis, we reveal that glutathionylation of Prx I, WT, or its C52/173S double mutant shifted its oligomeric status from decamers to a population consisting mainly of dimers. Cys83 is localized at the putative dimer-dimer interface, implying that the redox status of Cys83 may play an important role in stabilizing the oligomeric state of Prx I. Studies with the Prx I (C83S) mutant show that Cys83 is not essential for the formation of high molecular weight complexes. Glutathionylation of the C83S mutant leads to accumulation of dimers and monomers. In addition, glutathionylation of Prx I, both the WT and C52/173S mutants, greatly reduces their molecular chaperone activity in protecting citrate synthase from thermally induced aggregation. Together, these results reveal that glutathionylation of Prx I promotes changes in its quaternary structure from decamers to smaller oligomers, and concomitantly inactivates its molecular chaperone function. In addition, when living cells were treated with 10 to 50 M of hydrogen peroxide for 10 min and glutathionylation was monitored with biotinylated glutathione, we found that only the Cys83-containing Prx I was clearly glutathionylated. This observation indicates that glutathione may not be easily accessible to interact with Cys52 and Cys173 in the dimer, while Cys83 is the favored glutathionylation site. Furthermore, the fact that glutathionylation of Cys83 in the Prx I (C52/173S) mutant causes a drastic reduction in its chaperone activity indicates that glutathionylation specifically on the Cys83 residue is sufficient for regulating the chaperone activity of Prx I. (iii) Regulatory studies of phospholipase C-gamma (PLC-gamma). PLC catalyzes the generation of second messengers in response to cellular stimulation in mammalian cells. Its isozyme, PLC-gamma, plays an important role in growth factor or immunoreceptor signaling. Activation of PLC-gamma is mediated by tyrosine phosphorylation catalyzed by receptor-integrated or -associated tyrosine kinases. PLC-gamma possesses a large insertion sequence, consisting of a split PH domain flanking two SH2 domains and an SH3 domain, between the catalytic X and Y subdomains. This sequence plays a central role in the regulation of PLC-gamma. We have been studying this multidomain fragment (50 kDa) hoping to reveal the regulatory mechanism of PLC-gamma. This fragment contains eight Cys residues, one of which was found to be reactive to an alkylating agent at neutral pH. We are currently trying to identify this unique cysteine residue. It may have a role in the regulation of PLC-gamma mediated by a redox sensitive signal. In addition, we found that the two major phosphorylation sites in the fragment, Y783 (activation site) and Y775 (no known function), are good substrates for many tyrosine kinases. Among them, receptor tyrosine kinases preferentially phosphorylate Y783, while non-receptor tyrosine kinases phosphorylate Y775 more favorably and only minimally phosphorylate Y783. This observation and the fact that immunoreceptor stimulation is known to rely on non-receptor tyrosine kinases to activate PLC-gamma and Y783 phosphorylation suggest a possible participation of adaptor protein(s) to facilitate the phosphrylation of Y783 by these kinases. To this end, we found that a unique sequence in the C-terminus of the second SH2 domain plays an important role in binding of receptor tyrosine kinases, but not non-receptor tyrosine kinases, and phosphorylation of Y783. (iv) Mechanism for the cellular uptake of cell penetrating peptides (CPP). CPP are a class of short peptides with repeating sequences of positively charged amino acids. These peptides are capable of traversing cell membranes either alone or when conjugated to much larger proteins, DNA fragments, or peptides. The mechanism of the translocation process remains unknown. While various endocytic pathways have been implicated, recent experiments employing inhibition of known endocytic pathways have failed to prevent CPP translocation. Other simulation studies on planar lipid bilayers have shown the presence of large membrane pores, although it is not clear why such pores would prevent transport of other smaller molecules. We found that fluorescent-labeled Arg-9 was transported into small lipid vesicles, suggesting endocytic pathways may not be required, but does not preclude peptide-induced vasiculations as a transport pathway. Based on our results, it is plausible that thevarious mechanisms reported thus far may be at play in the translocation process and that what determines the predominant pathway may be the energetics and nature of the peptide/membrane interactions. We are currently using Giant Unilamellar Vesicles (GUVs) as model systems and have built an FCS (Fluorescence Correlation Spectroscopy) system to gain a detailed understanding of CPP-mediated pathway at the membrane level.